Tei Maki

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca2+ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca2+ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus.

Reversible hydrogel formation driven by protein-peptide-specific interaction and chondrocyte entrapment.

We developed a hydrogel self-assembling method driven by the interaction between recombinant tax-interactive protein-1 (TIP1) with the PDZ domain in a molecule, which is fused to each end of the triangular trimeric CutA protein (CutA-TIP1), and a PDZ domain-recognizable peptide which is covalently bound to each terminus of four-armed poly(ethylene glycol) (PDZ-peptide-PEG). Genetic manipulation based on molecular-dynamic simulation generated a cell-adhesive RGD tripeptidyl sequence in the CutA loop region [CutA(RGD)-TIP1]. Spontaneous viscoelastic hydrogel formation occurred when either CutA-TIP1- or CutA(RGD)-TIP1-containing buffer solution and PDZ-peptide-PEG-containing buffer solutions were stoichiometrically mixed. Dynamic viscoelasticity measurement revealed shear stress-dependent reversible-phase transformation: a spontaneous viscoelastic hydrogel was formed at low shear stress, but it was transformed into a sol at high shear stress. Upon the cessation of shear, hydrogel was restored. When chondrocytes were pre-mixed with one of these two components containing buffer solutions, the stoichiometric mixed solution was also spontaneously gelled. Individual rounded cells and multicellular aggregates were entrapped within both hydrogels without substantial cellular impairment regardless of the presence or absence of RGD motif in the CutA-TIP1 molecule. The potential use of such a shear-sensitive hydrogel for injectable cell delivery into diseased or lost cartilage tissue is discussed.

Nanoscale elongating control of the self‐assembled protein filament with the cysteine‐introduced building blocks

Self‐assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self‐assembling proteins, named “Nanolego.” Nanolego consists of “structural elements” of a structurally stable symmetrical homo‐oligomeric protein and “binding elements,” which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self‐assembly process. The stabilization method is mediated by disulfide bonds between Cysteine‐residues incorporated into the binding elements, and the termination method uses “capping Nanolegos,” in which some of the binding elements in the Nanolego are absent for the self‐assembled ends. With these technologies, we successfully constructed timing‐controlled and size‐regulated filament‐shape complexes via Nanolego self‐assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.

ELECTROLYZED AND NATURAL REDUCED WATER EXHIBIT INSULIN-LIKE ACTIVITY ON GLUCOSE UPTAKE INTO MUSCLE CELLS AND ADIPOCYTES

In the type 2 diabetes, it has become clear that reactive oxygen species (ROS) cause reduction of glucose uptake by inhibiting the insulin-signaling pathway in muscle cells and adipocytes. We demonstrated that electrolyzed-reduced water (ERW) scavenges ROS and protects DNA from oxidative damage1). Here we found that ERW scavenges ROS in insulin-responsive L6 myotubes and mouse3T3/L1 adipocytes. Uptake of 1-deoxy-D- glucose (2-DOG) into both L6 cells and 3T3/L1 cells was stimulated by ERW in the presence or absence of insulin. This insulin-like activity of ERW was mediated by the activation of PI-3 kinase, resulting in stimulation of translocation of glucose transporter GLUT4 from microsome to plasma membrane. These results suggest that ERW may be useful to improve insulin-independent type 2 diabetes.

Nanoscale elongating control of the self-assembled protein filament with the cysteine-introduced building blocks (Protein Science (2009) 18, (960-969))

CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), Tsurumi-ku, Yokohama 230-0045, Japan Genome Science Laboratory, RIKEN, Hirosawa, Wako 351-0198, Japan Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka 819-0395, Japan High-Performance Molecular Simulation Team, Computational Systems Biology Group, Advanced Computational Sciences Department, RIKEN Advanced Science Institute, Tsurumi-ku, Yokohama, Kanagawa 230-0046, Japan Genome Biotechnology Laboratory, Kanazawa Institute of Technology, Ishikawa 924-0838, Japan